Constitutive photomorphogenesis 1 (cop1) nucleic acid sequence from zea mays and its use thereof

ABSTRACT

The present invention relates to an isolated COP1 nucleic acid sequence from a maize plant and the isolated COP1 nucleic acid sequence is named as ZmCOP1. The present invention also relates to a method of using the ZmCOP1 nucleic acid sequence to control the shade avoidance response of a crop plant for high density farming and yield enhancement.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims benefit of under 35 USC 119(e) of U.S.provisional application Ser. No. 60/315,593 filed Aug. 29, 2001. ThisApplication is a divisional of the non-provisional application with Ser.No. 10/229,436 filed Aug. 28, 2002. Both of these Applications areherein incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of thesequence listing are provided. They contains 17 nucleotide and proteinsequences and are herein incorporated by reference. This sequencelisting is identical in content to the listing in the Ser. No.10/229,436 case which is also incorporated by reference.

INTRODUCTION

The present invention is in the field of plant molecular biology. Morespecifically the present invention relates to an isolated nucleic acidmolecule, a protein and fragments of the protein that the isolatednucleic acid molecule encodes. Most specifically, the present inventionrelates to a constitutive photomorphogenesis 1 (COP1) nucleic acidsequence from Zea mays that encodes a COP1 protein and fragments of theCOP1 protein associated with plant photomorphogenesis. The presentinvention also relates to a method of using the isolated COP1 nucleicacid molecules, the COP1 proteins and fragments of the COP1 proteins formolecular manipulation of shade avoidance responses of crop plants tolight for improving their density tolerance and thereafter for enhancingtheir yield when planted at a high population density.

BACKGROUND OF THE INVENTION

Plant growth is a highly malleable process that is strongly influencedby environmental factors, especially light. Light plays a vital role inplants' photomorphogenesis and affects almost all aspects of plantgrowth and development. The effects of light on plant development areespecially prominent at the seedling stage. Under normal lightconditions with unobstructed direct light, a plant seedling developsaccording to a characteristic photomorphogenic pattern, that is, it hasopen, expanded cotyledons and a short hypocotyl. This developmentalpattern rapidly establishes the seedling as a photoautotrophic organism,and most of the plant's energy is devoted to cotyledon and leafdevelopment while longitudinal extension growth is minimized. A seedlinggrowing in darkness, however, will etiolate, displaying elongatedhypocotyls and closed and unexpanded cotyledons. Under low lightconditions where light quality and intensity are reduced by shading,obstruction or high population density, a seedling develops according toa different pattern as a shade-avoiding seedling that displays reducedcotyledon expansion relative to the seedling grown in unobstructedlight, and hypocotyl extension is greatly increased. During thisdevelopmental response of the seedling to the low light conditions, thehypocotyl is elongated which couples with reduction in cotyledon andleaf expansion.

Thus, a significant problem for crop fainting is created when cropplants are grown at high population density as it often results in a lowlight level for each individual plant. To compete for this light, plantshave to re-distribute their energy and nutrition towards heightextension, often called a shade avoidance response, resulting in anaccelerated stem elongation and thin stems. This shade avoidanceresponse to poor light conditions in a populated environment oftenresults in crop yield loss. For example, in maize plants, accumulatingevidence suggests that the stem elongation process itself may be linkedto suppression of ear development. Corn prolificacy and earestablishment are sensitive to light intensity. High population densitymay cause abortion of ear development at lower nodes, even at all nodes.High density leads to most of the red and blue spectra of the sunlightbeing absorbed by the upper leaves, leaving the far-red light filteredor reflected to the lower canopy. The red/far-red ratio is a function ofcanopy density. If the density is high, the red/far-red ratio is low.This low ratio triggers the shade avoidance response, in which theplants distribute resources for stem elongation in a competition forsunlight (Quail et al, Science 268, 675-680, 1995). Reduction orelimination of the shade avoidance response has been shown to improveharvest index or yield (Maliakal et al, Critic. Rev. Plant Sci. 17,465-539,1999; Thiele et al, Plant Physiol. 120, 73-81, 1999; Robson etal, Nature Tech. 14, 995-998, 1996). Thus, the shade avoidance responseis relevant to the harvest index, for example at high populationdensity.

Various attempts have been made to overcome the shade avoidance problemin crop farming. Breeding efforts usually result in shorter plants and,in the case of corn, smaller tassels to save energy and nutrition forkernel development (Duvick and Cassman, Crop Sci. 39, 1622-1630, 1999;Chapman and Edmeades, Crop Sci. 39, 1315-1324, 1999). Molecular andbiotechnological approaches have also been tried to identify a gene or aset of genes that manipulate the photomorphogenesis pathway in a mannermodifying the plant architecture to have shorter internodes. Such aplant, when growing in a dense population, would have the ability torespond to low light environment without extending its stem, therebyminimizing the shade avoidance response and enhancing yield (see, forexample, Smith, U.S. Pat. No. 5,945,579; Hershey and Keller, U.S. Pat.No. 5,268,526; Deng et al., PCT Application WO00/18940).

In recent decades, many genes or gene mutants in light-signaltransduction and shade avoidance response pathways have been identifiedand studied (Chory, Plant Cell 9: 1225-1234, 1997; Chory et al., Cell58: 991-999, 1989; Deng et al., Genes Dev. 5: 1172-1182, 1991;Karlin-Neumann et al., Plant Physiol. 88: 1323-1331, 1988; Lissemore andQuail, Mol. Cell Biol. 8: 4840-4850, 1988; U.S. Pat. No. 5,945,579;McNellis and Deng, Plant Cell 7: 1749-1761, 1995; Nagatani et al., PlantPhysiol. 102: 269-277, 1993; Osterlund et al., Trends Cell Bio. 9:113-118, 1999; Parks and Quail, Plant Cell 5: 39-48, 1993). Among thesegenes, a constitutive photomorphogenesis 1 gene (COP1) from Arabidopsishas been studied and demonstrated to be regulated by light during plantdevelopment in response to different light conditions (Osterlund et al.,Trends Cell Bio. 9: 113-118, 1999; Deng et al., Cell 71: 791-801, 1992;McNellis et al., Plant Cell 6: 1391-1400, 1995; McNellis et al., PlantCell 8: 1491-1503, 1996; Osterlund and Deng, Plant Journal 16 (2):201-208, 1998; Stacey et al., Plant Cell 11: 349-363; Torii et al., EMBO17: 5577-5587, 1998; von Arnim and Deng, Cell 79: 1035-1045; Yamamoto etal., Plant Cell 10: 1083-1094, 1998; Deng et al., PCT ApplicationWO00/18940). The COP1 gene was initially identified through recessiveloss-of-function mutations in Arabidopsis that display a constitutivelyphotomorphogenic phenotype regardless of light conditions (Deng et al.,Genes Dev. 5: 1172-1182, 1991). The constitutively photomorphogeniephenotype and recessive nature of cop1 mutations indicate that COP1 mayact as a negative regulator, or light-inactivated repressor, ofphotomorphogenesis. The COP1 gene in Arabidopsis encodes a protein thatcontains three recognizable domains: a ring finger domain (zinc-bindingmotif), a coiled-coil domain and multiple WD-40 repeats characteristicof the B subunit of trimeric G-proteins (Deng et al., Cell 71: 791-801,1992; PCT Application WO00/18940). These protein domains have beenimplicated in protein-protein interactions, and thus COP1 might interactwith multiple partners via these interactive domains to regulate plantmorphogenic development and the shade avoidance response. Overexpressionof a full-length COP1 results in quantitative hypersuppression ofphotomorphogenic development (McNellis et al., Plant Cell 6: 1391-1400,1995), which suggests that COP1 plays a role in a regulatory step inmediating the repression of photomorphogenic development (Osterlund etal., Trends Cell Bio. 9: 113-118, 1999; Deng et al., PCT Application WO00/18940). The wild-type COP1 protein normally acts to repress thephotomorphogenic pathway in darkness and light reverses this repression.COP1 appears to be a downstream light-signaling component (Deng et al.,Cell 71: 791-801, 1992; PCT Application WO 00/18940). Overexpression ofa fragment of COP1 in Arabidopsis is hypothesized to down regulatenative COP1, this has also resulted in shorter stems of transgenicplants growing under low light conditions in comparison with those ofwild-type plants (see, Deng et al., PCT Application WO 0018940).

Thus, the COP1 proteins in plants growing at low light conditions suchas in a highly populated environment will act to repress normalphotomorphogenic development of these plants and help activate shadeavoidance response pathway to stimulate stem elongation. Therefore,reducing the level of functional COP1 proteins in plants might produce aphenotype typical of plants growing at high light intensity conditionseven when the plants are under low light conditions. This phenotypecould include well developed leaves, more chloroplasts, shorter andthicker stems.

Although some studies have been done to understand the role of COP1proteins in plant morphogenesis and development, there is littlereported effort on utilizing COP1 to deal with an unsolved, commonproblem in crop farming; that is the shade avoidance response of plants.Deng and his colleagues (Deng et al, PCT application WO 00/18940)disclosed an isolated COP1 nucleic acid from Arabidopsis and use of saidCOP1 Their publication was directed to improved seedling emergencecharacteristics and not to a solution to shade avoidance relatedproblems in crop plants grown at high population density.

Thus, there exists a need in the field for a new and different approachto reduce or diminish the shade avoiding response of crop plants growingat high population density. There exists a need, through use of adifferent light transduction component, i.e., COP1 gene, to improve someof crop plants' agronomic traits such as reduced stem length andincreased shade tolerance that are closely associated with crop yield.

SUMMARY OF THE INVENTION

Therefore, the present invention, in one aspect, relates to an isolatednucleic acid molecule from a maize plant (Zea mays) comprising afull-length nucleic acid sequence from a cDNA identified as ZmCOP1 andhaving the function of improving crop plants' agronomic traits that areassociated with the crop yield. ZmCOP1 comprises 2230 nucleotides codinga polypeptide with 693 amino acid residues. The sequence of ZmCOP1comprises SEQ ID NO: 12.

The present invention, in another aspect, provides an isolated nucleicacid from Zea mays comprising a nucleotide sequence, wherein thenucleotide sequence is defined as follows: (1) the nucleotide sequencehas at least 80% sequence identity to a sequence comprising SEQ ID NO:12; (2) the nucleotide sequence hybridizes under stringent conditions tothe complement of a second isolated nucleic acid, wherein the nucleotidesequence of the second isolated nucleic acid comprising SEQ ID NO: 12;or (3) the nucleotide sequence is complementary to (1) or (2).

The present invention, in still another aspect, provides an isolatednucleic acid from Zea mays comprising a nucleotide sequence, wherein thenucleotide sequence is defined as follows: (1) the nucleotide sequenceencodes a polypeptide having an amino acid sequence that has at least90% sequence identity to a sequence comprising SEQ ID NO: 13; (2) thenucleotide sequence hybridizes under stringent conditions to thecomplement of a second isolated nucleic acid, wherein the nucleotidesequence of the second isolated nucleic acid encodes a polypeptidehaving an amino acid sequence comprising SEQ ID NO: 13; or (3) thenucleotide sequence is complementary to (1) or (2).

The present invention, in yet another aspect, also relates to arecombinant DNA construct for producing high-density tolerant cropplants. The construct comprises a light inducible promoter, a COP1structural nucleic acid sequence that comprises a sequence at least 80%identical to SEQ ID NO: 12 or a fragment thereof, and a transcriptionterminator. The recombinant DNA construct causes reduction of theindigenous COP1 protein level upon its transformation into a crop plantthrough introduction of the COP1 structural nucleic acid sequence in anantisense orientation wherein an antisense COP1 mRNA is transcribed andbase-paired with the indigenous COP1 mRNA. The recombinant DNA constructalso causes the reduction of the indigenous COP1 protein level upon itstransformation into a crop plant by overexpressing a full length or afragment of the COP1 protein that binds to a native COP1 protein andmakes the COP1 protein complex non-functional. With the reduction of thenative COP1 protein level in the crop plants the density tolerance ofthe crop plants is improved and the crop plants may be overplanted at ahigh population density to achieve enhanced yield.

The light inducible promoter used in the recombinant DNA construct maybe, but may not be limited to, a cab promoter, an ATHB-2 promoter, or afar-red light inducible promoter for the antisense approach oroverexpression of the COP1 nucleic acid sequence or a fragment thereof.

The present invention, in yet another aspect, also relates to transgeniccrop plants that demonstrate a high-density tolerant trait. Thesetransgenic crop plants contain exogenous COP1 nucleic acid sequencesthat may be in an “antisense” orientation or may be overexpressed. Theexogenous COP1 nucleic acid sequences are at least 80% identical to SEQID NO: 12 or fragments thereof. In a preferred embodiment, the cropplants contain a full-length ZmCOP1 nucleic acid sequence having SEQ IDNO: 12 or a fragment thereof. In one example of the present inventionwhen a fragment is considered, the fragment contains about 1233nucleotides from the 5′ end of the ZmCOP1 having SEQ ID NO: 14. Inanother example of the present invention, the fragment contains about906 nucleotides from the 5′ end having SEQ ID NO: 16. The transgeniccrop plants have reduced levels of the native COP1 proteins from theirnative COP1 nucleic acid sequences. The transgenic crop plants alsodemonstrate a number of other desirable agronomic traits over wild-typecrop plants in that they have shorter stems and more sturdyarchitecture.

The present invention, in yet still another aspect, also provides amethod of overplanting crop plants at a high population density foryield enhancement by producing the transgenic crop plants with reducedCOP1 protein level in comparison to that of the wild crop plants.Through reduction of the COP1 protein levels in the transgenic cropplants the architecture of the transgenic plants is modified and theirshade avoidance responses to light are minimized. In a preferredembodiment, the levels of the functional endogenous COP1 proteins in thetransgenic crop plants may be reduced by binding an endogenous COP1 mRNAwith an antisense ZmCOP1 sequence that comprises the full-length ZmCOP1nucleic acid sequence of the present invention encoding SEQ ID NO: 13.The levels of the functional endogenous COP1 proteins in the transgenicplants may also be reduced by binding the endogenous COP1 mRNA with anantisense ZmCOP1 sequence that only comprises a fragment of the COP1nucleic acid sequence. The level of the functional endogenous COP1proteins may also be reduced by overexpressing a full length unalteredor mutated ZmCOP1 protein or a fragment thereof with binding domainsthat binds to a native endogenous COP1 protein and thus rendering theendogenous COP1 protein complex non-functional. The fragment of theZmCOP1 protein used in the present invention as an example may comprise411 amino acid residues from the N terminal end having SEQ ID NO: 15that comprises a protein-binding domain. The fragment of the COP1protein in another example may also comprise 301 amino acid residuesfrom the N terminal end having SEQ ID NO: 17 that comprises aprotein-binding domain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. Nucleotide sequence alignment of Arabidopsis COP1(AtCOP1_gi-402684) and maize (Zea mays) COP1 (ZmCOP1). The homologycomparison indicates that these two nucleotide sequences have a 50%sequence identity.

FIG. 2A-D. Nucleotide sequence alignment of the rice (Oryza sativa) COP1(OsCOP1, gi7592844) and maize (Zea mays) COP1 (ZmCOP1). The homologycomparison indicates that these two nucleotide sequences have a 77%sequence identity.

FIG. 3A-B. Peptide sequence alignment of maize (Zea mays) COP1 (ZmCOP1)and selected COP1 peptide sequences from other plants. ZmCOP1 is frommaize (Zea mays), PsCOP1 from pea (Pisum sativum), At COP1 fromArabidopsis thaliana, COP1 from Japanese morning glory (Ipomoea nil),OsCOP1 from rice (Oryza sativa), and LeCOP1 from tomato (Lycopersiconesculentum).

FIG. 4. A plasmid map of pMON47119. The coding sequence of theN-terminal end 411 amino acid residues of ZmCOP1 was placed under thecontrol of a cab promoter.

FIG. 5. A plasmid map of pMON47118. The coding sequence of theN-terminal end 301 amino acid residues of ZmCOP1 was placed under thecontrol of a cab promoter.

FIG. 6. A plasmid map of pMON47120. ZmCOP1 was cloned in the vector inreverse orientation and placed under the control of a cab promoter.

FIG. 7. A plasmid map of pMON47130. The coding sequence of theN-terminal end 301 amino acid residues of ZmCOP1 was placed under thecontrol of a rice-actin promoter (RACT) promoter.

FIG. 8. A plasmid map of pMON47131. The coding sequence of the fulllength ZmCOP1 was cloned in the vector in reverse orientation and wasplaced under the control of a rice-actin promoter (RACT) promoter.

FIG. 9. The height comparison of the transformed R1 plants with the wildtype plants at maturity. The average height and growth rate of some R1transformed plants from pMON47118 in several events were lower comparedto those of the wild type plants growing nearby. LH172 is a wild typeinbred used for transformation; ZM is a transformed R1 plant; LH172/ZMrepresents a F1 plant obtained by crossing LH172 plant with atransformed R0 plant. Each number under each bar in the figure, e.g., ZM535321, represents one event.

FIG. 10. Western analysis of YAA plants

FIG. 11. Plant height of some YAA events at V11 stage

FIG. 12. Western analysis of Kyle plants grown under normal (full greenhouse) light and weak light.

FIG. 13. Western blot analysis to determine protein expression levels inKyle 17 and Kyle 50 events. Both Kyle positive (Pos) and negative (Neg)events are shown at different growth stages. Stage 1 represents theV3-V4 stage, 2 represents the V5-V7 stage, and 3 the VT stage. P is apositive control.

FIG. 14. Mesocotyl lengths of Kyle seedlings grown under 1 micromole permeter squared of light.

FIG. 15. Plant height of 5 Kyle events four weeks after transplanting.

FIG. 16. Picture of positive (on both sides) and negative (center) linesfrom the Kyle 77 event.

FIG. 17. Height of plants from the Kurt R1/F1 plants grown in the field.Shown below is the expression level of their transgene by Westernanalysis.

DETAILED DESCRIPTION

Provided below are the following definitions to aid those skilled in theart in understanding the detailed description of the present invention.

As used herein, “antisense technology” refers to a method to introduceinto cells a RNA or single-stranded DNA molecule that is complementaryto the mRNA of the target gene. This antisense molecule may base-pairwith the endogenous mRNA, preventing translation of the mRNA into aprotein.

As used herein, a “coding sequence”, “structural nucleotide sequence” or“structural gene” is a nucleotide sequence that is translated into apolypeptide, usually via mRNA, when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a translation start codon at the 5′-terminus and atranslation stop codon at the 3′-terminus. A coding sequence mayinclude, but may not be limited to, genomic DNA, cDNA, and recombinantnucleotide sequences.

As used herein, a constitutive photomorphogenesis 1 nucleic acid, or“COP1 nucleic acid”, refers to a nucleic acid encoding all or part of aspecific constitutive photomorphogenesis 1 protein, or “COP1 protein”. ACOP1 nucleic acid may be defined functionally by its ability to confer amodulated photomorphogenic response upon transformation into a plant.The COP1 nucleic acids may include any COP1 nucleic acids from anysource. The exemplary COP1 nucleic acid is the COP1 nucleic acid asdisclosed in the present invention.

As used herein, a “C-terminal region” refers to the region of a peptide,polypeptide, or protein chain from the middle thereof to the end thatcarries the amino acid having a free carboxyl group. A “N-terminalregion” refers to the region of a peptide, polypeptide, or protein chainfrom the amino acid having a free amino group to the middle of thechain.

As used herein, “expression” refers to the transcription and stableaccumulation of sense (mRNA) or antisense RNA derived from the nucleicacid of the invention. Expression may also refer to translation of mRNAinto a polypeptide. Also as used herein, “overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms.

As used herein, a “genotype” refers to the genetic constitution, latentor expressed, of a plant, the sum total of all genes present in anindividual. As used herein, a “phenotype” of a plant is any of one ormore characteristics of a plant (e.g. male sterility, yield, qualityimprovements, etc.), as contrasted with the genotype. A change ingenotype or phenotype may be transient or permanent.

As used herein, a “homolog” of a nucleotide sequence refers to anisolated nucleic acid sequence which is substantially the same as theCOP1 nucleic acid sequence of the present invention or its complementarynucleotide sequence. A “homolog” of the COP1 nucleic acid sequence is apolynucleotide sequence from a plant species that encodes a polypeptidethat is functionally similar to COP1 and that preferably has substantialamino acid sequence identity or similarity to COP1 from maize.

Planting or population density varies from a crop to a crop, from agrowing region to another region and from a year to another year. Asused herein, the term “high population density” is defined as a densityat least 10% to 100% higher than the average prevailing density for agiven crop in a given growing region. Preferably, the high populationdensity is at least 10% higher, more preferably at least 40% higher,more preferably at least 70% higher, and most preferably at least 100%higher than the average prevailing density for the given crop in thegiven growing region. The “average prevailing density” is defined as theaverage of the planting density used by the majority of farmers in aregion. Taken corn as an example, the average prevailing density is20,000 plants per acre in Missouri, USA. The higher population densityis preferably at least 22,000 plants per acre, more preferably at least28,000 plants per acre, more preferably at least 34,000 plants per acre,and most preferably at least 40,000 plants per acre.

The average prevailing densities of a few crop plants in the USA in 2000are listed below (Table 1). The examplery crop species are just examplesand, therefore, may not be construed as limitations to the scope of thepresent invention. Similarly, the country selected above, i.e., USA, isalso an example in which the average prevailing densities of these fewcrop plants can be demonstrated. It may not be construed as a limitationof the present invention.

TABLE 1 The average prevailing densities of a few crop plants in the USA(per acre) Crop Name Density Corn 20,000-25,000 Wheat1,000,000-1,500,000 Rice 650,000-900,000 soybean 150,000-200,000 Canola260,000-350,000 Sunflower 17,000-23,000 Cotton 28,000-55,000

As used herein, “hybridization” refers to the ability of a strand ofnucleic acid to join with a complementary strand via base pairing.Hybridization occurs when complementary sequences in the two nucleicacid strands bind to one another.

As used herein, “identical” nucleotide or protein sequences aredetermined by using programs such as a BLAST program (Altschul et al.,Nucleic Acids Res. 25:3389-3402; 1997) using the default parameters(Expectation value (E): blank; Alignment view options: pairwise; Filterquery sequence: no; Cost to open a gap: 0; Cost to extend a gap: 0; Xdropoff value for gapped alignment: 0; Show GI's in deflines: no;Penalty for a nucleotide mismatch: −3; Reward for a nucleotide match: 1;Threshold for extending hits: 0; Perform gapped alignment: yes; QueryGenetic code to use: standard; DB Genetic code: standard; Believe thequery define: no; Matrix: BLOSUM62; Word size: 0; Effective length ofthe database: 0; Query strand Use: both).

As used herein, an “isolated” nucleic acid is one that has beensubstantially separated or purified away from other nucleic acidsequences in the cell of the organism in which the nucleic acidnaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA, by conventional nucleic acid-purification methods. The term alsoembraces recombinant nucleic acids and chemically synthesized nucleicacids.

The term “polypeptide” or “protein”, as used herein, refers to a polymercomposed of amino acids connected by peptide bonds. The term“polypeptide” or “protein” also applies to any amino acid polymers inwhich one or more amino acid residue is an artificial chemical analogueof a corresponding naturally occurring amino acid, as well as to anynaturally occurring amino acid polymers. The essential nature of suchanalogues of naturally occurring amino acids is that, when incorporatedinto a protein, that protein is specifically reactive to antibodieselicited to the same protein but consisting entirely of naturallyoccurring amino acids. It is well known in the art that proteins orpolypeptides may undergo modification, including but not limited to,disulfide bond formation, gamma-carboxylation of glutamic acid residues,glycosylation, lipid attachment, phosphorylation, oligomerization,hydroxylation and ADP-ribosylation. Exemplary modifications aredescribed in most basic texts, such as, for example, Proteins—Structureand Molecular Properties, 2nd ed. (Creighton, Freeman and Company, N.Y., 1993). Many detailed reviews are available on this subject, such as,for example, those provided by Wold (In: Post-translational CovalentModification of Proteins, Johnson, Academic Press, N. Y., pp. 1-12,1983), Seifter et al. (Meth. Enzymol. 182: 626, 1990) and Rattan et al.(Ann. N.Y. Acad. Sci. 663: 48-62, 1992). Modifications can occuranywhere in a polypeptide, including the peptide backbone, the aminoacid side chains and the amino or carboxyl termini. In fact, blockage ofthe amino or carboxyl group in a polypeptide, or both, by a covalentmodification, is common in naturally occurring and syntheticpolypeptides and such modifications may be present in polypeptides ofthe present invention, as well. For instance, the amino terminal residueof polypeptides made in E. coli or other cells, prior to proteolyticprocessing, almost invariably will be N-formylmethionine. Duringpost-translational modification of the polypeptide, a methionine residueat the NH₂ terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine containing and themethionine-less amino terminal variants of the protein of the invention.Thus, as used herein, the term “protein” or “polypeptide” includes anyprotein or polypeptide that is modified by any biological ornon-biological process. The terms “amino acid” and “amino acids” referto all naturally occurring amino acids and, unless otherwise limited,known analogs of natural amino acids that can function in a similarmanner as naturally occurring amino acids.

As used herein, the term “isolated polypeptide” refers primarily to apolypeptide produced by expression of an isolated nucleic acid moleculeof the present invention or by chemically synthesizing process.Alternatively, this term may refer to a polypeptide which has beensufficiently separated from other polypeptides or proteins with which itwould naturally be associated, so as to exist in substantially pureform. Also as used herein, a “functionally equivalent fragment” of theisolated polypeptide refers to a polypeptide that lacks at least oneresidue a native full length COP1 polypeptide. Such a fragment retainsCOP1 activity when expressed in a transgenic plant or possesses acharacteristic functional domain or an immunological determinantcharacteristic of a native COP1 polypeptide. Immunologically activefragments typically have a minimum size of 7 or 17 or more amino acids.Preferably, COP1 fragments are at least 10 amino acids in length.

As used herein, the term “native” refers to a naturally occurring (“wildtype”) nucleic acid or polypeptide.

As used herein, a “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. The percentage of sequence identity may be determined by usingprograms such as a BLAST program (Altschul et al., Nucleic Acids Res.25:3389-3402; 1997) using the default parameters.

As used herein, a “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence. Thepromoter sequence consists of proximal and more distal upstreamelements, the later elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence which can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic DNA segments. Itis understood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a gene to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. Promoters which cause conditional expressionof a structural nucleotide sequence under the influence of changingenvironmental conditions or developmental conditions are commonlyreferred to as “inducible promoter”.

“Promoter” refers to a DNA sequence that binds an RNA polymerase (andoften other transcription factors as well) and promotes transcription ofa downstream DNA sequence. Said sequence can be an RNA that hasfunction, such as rRNA (ribosomal RNA) or tRNA (transfer RNA). Often,the RNA produced is a hetero-nuclear (hn) RNA that has introns which arespliced out to produce an mRNA (messenger RNA). A “plant promoter” is anative or non-native promoter that is functional in plant cells.Constitutive promoters are functional in most or all tissues of a plantthroughout plant development. Tissue-, organ- or cell-specific promotersare expressed only or predominantly in a particular tissue, organ, orcell type, respectively “Specifically” expressed and “enhanced”expression are not distinguishable and are used inter-changeably herein.Often, a promoter discussed as “specifically” expressed in one paper orpatent is found to only offer “enhanced” expression in that tissue asthe number of tissues studied for expression is increased, or moresensitive techniques are used to study expression in the same tissues.“Enhanced expression” is used herein to refer to any promoter thatprovides an increased expression in a single tissue or developmentalstage, or under a particular environmental condition, but causesexpression, even significant expression, in other tissue(s), ordevelopmental stage(s), or environmental condition(s).

Temporally regulated promoters are functional only or predominantlyduring certain periods of plant development or at certain times of day,as in the case of genes associated with circadian rhythm, for example.Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding, cold, or chemicals such as ethanol, jasmonate, salicylic acid,or safeners.

Any plant promoter can be used as a 5′ regulatory sequence formodulation expression of a particular gene or genes. One preferredpromoter would be a plant RNA polymerase II promoter. Plant RNApolymerase II promoters, like those of other higher eukaryotes, havecomplex structures and are comprised of several distinct elements. Onesuch element is the TATA box or Goldberg-Hogness box, which is requiredfor correct expression of eukaryotic genes in vitro and accurate,efficient initiation of transcription in vivo. The TATA box is typicallypositioned at approximately −25 to −35, that is, at 25 to 35 basepairs(bp) upstream (5′) of the transcription initiation site, or cap site,which is defined as position+1 (Breathnach and Chambon, Ann. Rev.Biochem. 50:349-383, 1981; Messing et al., In: Genetic Engineering ofPlants, Kosuge et al., eds., pp. 211-227, 1983). Another common element,the CCAAT box, is located between −70 and −100 bp. In plants, the CCAATbox may have a different consensus sequence than the functionallyanalogous sequence of mammalian promoters (the plant analogue has beenteinied the “AGGA box” to differentiate it from its animal counterpart;Messing et al., In: Genetic Engineering of Plants, Kosuge et al., eds.,pp. 211-227, 1983). In addition, virtually all promoters includeadditional upstream activating sequences or enhancers (Benoist andChambon, nature 290:304-310, 1981; Gruss et al., Proc. Nat. Acad. Sci.USA 78:943-947, 1981; and Khoury and Gruss, Cell 27:313-314, 1983)extending from around 100 bp to 1,000 bp or more upstream of thetranscription initiation site.

When fused to heterologous DNA sequences, such promoters typically causethe fused sequence to be transcribed in a manner that is similar to thatof the gene sequence with which the promoter is normally associated.Promoter fragments that include regulatory sequences can be added (forexample, fused to the 5′ end of, or inserted within, an active promoterhaving its own partial or complete regulatory sequences (Fluhr, et al.,Science 232:1106-1112, 1986; Ellis et al., EMBO J. 6:11-16, 1987;Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987;Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., PlantMol. Biol. 15:373-381, 1991). Alternatively, heterologous regulatorysequences can be added to the 5′ upstream region of an inactive,truncated promoter, e.g., a promoter including only the core TATA and,sometimes, the CCAAT elements (Fluhr, et al., Science 232:1106-1112,1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990,1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).

Promoters are typically comprised of multiple distinct “cis-actingtranscriptional regulatory elements,” or simply “cis-elements,” each ofwhich can confer a different aspect of the overall control of geneexpression (Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987; Benfey et al.,EMBO J. 9:1677-1684, 1990). “cis elements” bind trans-acting proteinfactors that regulate transcription. Some cis elements bind more thanone factor, and trans-acting transcription factors may interact withdifferent affinities with more than one cis element (Johnson andMcKnight, Ann. Rev. Biochem. 58:799-839, 1989). Plant transcriptionfactors, corresponding cis elements, and analysis of their interactionare discussed, for example, in: Martin, Curr. Opinions Biotech.7:130-138, 1996; Murai, In: Methods in Plant Biochemistry and MolecularBiology, Dashek, ed., CRC Press, 1997, pp. 397-422; and Methods in PlantMolecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995,pp. 233-300. The promoter sequences of the present invention can contain“cis elements” which can modulate gene expression. Cis elements can bepart of the promoter, or can be upstream or downstream of said promoter.Cis elements (or groups thereof) acting at a distance from a promoterare often referred to as repressors or enhancers. Enhancers act toupregulate the transcriptional initiation rate of RNA polymerase at apromoter, repressors act to decrease said rate. In some cases the sameelements can be found in a promoter and an enhancer or repressor. Ciselements are generally sites where transcription factors bind to the DNAand modulate the rate at which RNA polymerase binds to the promoter.

The term “constitutive promoter” means a regulatory sequence that causesexpression of a structural nucleotide sequence in most cells or tissuesat most times. Constitutive promoters are active under manyenvironmental conditions and states of development or celldifferentiation. A variety of constitutive promoters are well known inthe art. Examples of constitutive promoters that are active in plantcells include but are not limited to the nopaline synthase (NOS)promoters; the cauliflower mosaic virus (CaMV) 19S and 35S (sometimescalled 35S herein, or a derivative of which is called e35S {U.S. Pat.Nos. 5,359,142, 5,196,525, 5,322,938, 5,164,316, and 5,424,200}); thetobacco mosaic virus promoter; the figwort mosaic virus promoters; andactin promoters, such as the Arabidopsis actin gene promoter (see, e.g.,Huang et al., Plant Mol. Biol. 33:125-139 (1997).

The term “tissue-specific promoter” means a regulatory sequence thatcauses an enhancement of transcription from a downstream gene inspecific cells or tissues at specific times during plant development,such as in vegetative tissues or reproductive tissues. Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only (or primarily only) in certain tissues,such as vegetative tissues, e.g., roots, leaves or stems, orreproductive tissues, such as fruit, ovules, seeds, pollen, pistols,flowers, or any embryonic tissue. Reproductive tissue specific promotersmay be, e.g., ovule-specific, embryo-specific, endosperm-specific,integument-specific, seed coat-specific, pollen-specific,petal-specific, sepal-specific, or some combination thereof. One skilledin the art will recognize that a tissue-specific promoter may driveexpression of operably linked sequences in tissues other than the targettissue. Thus, as used herein a tissue-specific promoter is one thatdrives expression preferentially in the target tissue, but may also leadto expression in other tissues as well.

Suitable seed-specific (inclusive of seed enhanced promoters) can bederived from the following genes: MAC1 from maize (Sheridan et al.,Genetics 142:1009-1020 (1996); Cat3 from maize (GenBank No. L05934,Abler et al., Plant Mol. Biol. 22:10131-1038 (1993); vivparous-1 fromArabidopsis (Genbank No. U93215); Atimycl from Arabidopsis (Urao et al.,Plant Mol. Biol. 32:571-57 (1996); Conceicao et al., Plant 5:493-505(1994), herein incorporated by reference in their entireties); napA fromBrassica napus (GenBank No. J02798); the napin gene family from Brassicanapus (Sjodahl et al., Planta 197:264-271 (1995)). Seed specificpromoters are an integral part of the current invention. It should benoted that a seed specific promoter can often cause the expression of agene in more than just seeds, or in more than one portion or tissue of aseed. Thus seed specific can be read as seed enhanced and is meant to beinclusive of any promoter that preferentially drives expression in anytissue in seed.

Promoters derived from genes encoding embryonic storage proteins, whichincludes the gene encoding the 2S storage protein from Brassica napus(Dasgupta et al, Gene 133:301-302 (1993); the 2s seed storage proteingene family from Arabidopsis; the gene encoding oleosin 20 kD fromBrassica napus (GenBank No. M63985); the genes encoding oleosin A(GenBank No. U09118) and oleosin B (GenBank No. U09119) from soybean;the gene encoding oleosin from Arabidopsis (GenBank No. Z17657); thegene encoding oleosin 18 kD from maize (GenBank No. J05212, Lee, PlantMol. Biol. 26:1981-1987 (1994)); and the gene encoding low molecularweight sulphur rich protein from soybean (Choi et al., Mol. Gen. Genet.246:266-268 (1995)), can also be used.

Promoters can also be induceable under particular environmentalconditions. For example a promoter could be upregulated, or even turnedon, by far-red light, cold, heat, drought, blue light (or any other mixof wavelengths), day length, or myriad other environmental conditions.These promoters could be isolated by the use of general molecularbiology techniques including transcription profiling of possible genes,and then isolation of the promoters of those genes through cloning andPCR.

As noted above, the present invention provides a recombinant DNAconstruct or expression vector that facilitates the expression of theCOP1 nucleic acid sequence discussed herein in plants. As used herein,the term “recombinant DNA construct” refer to assemblies of DNAfragments through genetic engineering operatively linked in a functionalmanner that direct the expression of the COP1 nucleic acid sequencediscussed herein, as well as any additional sequence(s) or gene(s) ofinterest in the plants.

As used herein, “regeneration” refers to the process of growing a plantfrom a plant cell or tissue (e.g., plant protoplast or explant).

As used herein, “sequence homology” refers to nucleic acid orpolypeptide sequence that has certain percentage of nucleotide or aminoacid similarity, as used in the present invention, to a native COP1nucleic acid or polypeptide sequence or COP1 nucleic acid or polypeptidesequence. Ordinarily, if a COP1 nucleic acid or polypeptide sequenceencompassed by the present invention has at least about 70% nucleotideor amino acid similarity to a native COP1 nucleic acid or polypeptidesequence or to a COP1 nucleic acid, preferably at least 80%, morepreferably at least about 90%, and most preferably at least about 95%similarity, such sequence homology is considered to be substantialhomology.

As used herein, the term “sequence identity” refers to amino acid ornucleic acid sequences that when compared using the local homologyalgorithm of Smith and Waterman in the BestFit program (WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis.,1981) are exactly alike.

As used herein, the term “sequence similarity” refers to amino acidsequences that when compared using the local homology algorithm of Smithand Waterman in the BestFit program (Wisconsin Package Version 10.0,Genetics Computer Group (GCG), Madison, Wis., 1981) match whenconservative amino acid substitutions are considered.

As used herein, “shade avoidance responses” refer to plants that, whengrowing at a high density condition or other shading environments, willcompete for light by elongating their stems unlimitedly. These plantswill usually be taller with thinner stems and have reducedphotosynthesis rate and reduced allocation of resource to fruits.

As used herein, a “stringent condition” is functionally defined withregard to hybridization of a nucleic-acid probe to a target nucleic acid(i.e., to a particular nucleic acid sequence of interest) by thespecific hybridization procedure discussed in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Hobart, 1989, at 9.52-9.55). Regarding the amplification of atarget nucleic acid sequence (e.g., by PCR) using a particularamplification primer pair, “stringent conditions” are conditions thatpermit the primer pair to hybridize substantially only to the targetnucleic acid sequence to which a primer having the correspondingwild-type sequence (or its complement) would bind so as to produce aunique amplification product. For hybridization of a probe or primer toa polynucleotide of another plant species in order to identify homologs,preferred hybridization and washing conditions are as discussed inSambrook et al. (supra, at 9.47-9.57, wherein “high stringentconditions” include hybridization at 65° C. in a hybridization solutionthat includes 6×SSC and washing for 1 hour at 65° C. in a wash solutionthat include 0.5×SSC, 0.5% SDS. “Moderate stringency” conditions aresimilar except that the temperature for the hybridization and washingsteps are performed at a lower temperature at which the probe isspecific for a target sequence, preferably at least 42° C., morepreferably at least 50° C., more preferably at least 55° C., and morepreferably at least 60° C. As used herein, a “tissue sample” is anysample that comprises more than one cell. In a preferred aspect, atissue sample comprises cells that share a common characteristic (e.g.,derived from a leaf, an ear or a stem, etc.).

As used herein, a “3′ untranslated region” or “3′ untranslated nucleicacid sequence” refers to a piece of transcribed but untranslated nucleicacid sequence at the 3′ end that functions in a plant cell to causetranscriptional termination and the addition of polyadenylatenucleotides to the 3′ end of said RNA sequence. Typically, a DNAsequence located from four to a few hundred base pairs downstream of thepolyadenylation site serves to terminate transcription. The region isrequired for efficient polyadenylation of transcribed messenger RNA(mRNA). RNA polymerase transcribes a coding DNA sequence through a sitewhere polyadenylation occurs.

As used herein, “transformation” refers to the transfer of a nucleicacid fragment into the genome of a host organism such as a host plant,resulting in genetically stable inheritance. Host plants containing thetransformed nucleic acid fragments are referred to as “transgenicplants”.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. Standard recombinantDNA and molecular cloning techniques used herein are well known in theart and are described in detail in Sambrook et al. (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1989).

Constitutive Photomorphogenesis 1 (COP1) Gene and Protein

The present invention is directed to an isolated constitutivephotomorphogenesis 1 (COP1) nucleic acid that encodes a COP1 protein. Asdisclosed in the present invention, the COP1 nucleic acid disclosedherein is isolated from a maize plant and is a full length COP1 cDNAsequence comprising 2230 nucleotides. Its COP1 protein comprises 693amino acid residues.

In a preferred embodiment, an isolated nucleic acid molecule of thepresent invention may comprise a nucleotide sequence or complementthereof, wherein the nucleotide sequence encodes a polypeptide having anamino acid sequence that has at least 90% sequence identity to SEQ IDNO: 13.

In a further preferred embodiment, an isolated nucleic acid molecule ofthe present invention may comprise a nucleotide sequence or complementthereof, wherein the nucleotide sequence encodes a polypeptide having anamino acid sequence that has at least 93% sequence identity to SEQ IDNO: 13.

In a more preferred embodiment, an isolated nucleic acid molecule of thepresent invention may comprise a nucleotide sequence or complementthereof, wherein the nucleotide sequence encodes a polypeptide having anamino acid sequence that has at least 96% sequence identity to SEQ IDNO: 13.

In a most preferred embodiment, an isolated nucleic acid molecule of thepresent invention may comprise a nucleotide sequence or complementthereof, wherein the nucleotide sequence encodes a polypeptide having anamino acid sequence that has at least 98% sequence identity to SEQ IDNO: 13.

The isolated nucleic acid of the present invention may also comprise anucleotide sequence or complement thereof, wherein the nucleotidesequence encodes a polypeptide having an amino acid sequence set forthin SEQ ID NO: 13 with conservative amino acid substitutions.

The present invention is directed to a method for manipulating COP1 geneexpression in transgenic plants to overcome shade avoidance responseswhen they grow in a highly populated environment. For this purpose, theCOP1 nucleic acid used in the present invention is not necessarily themaize COP1 nucleic acid disclosed herein. It can be any COP1 nucleicacids available in the art and these COP1 nucleic acids may include thesequences from Arabidopsis (Deng et al., Cell 27, 791-801, 1992), rice(gi7592844), tomato (gi4090943), pea (Zhao et al., Biochimica etBiophysica Acta-Gene Structure and Expression 1395, 326-328, 1998) andJapanese morning glory (Ipomoea nil). The species provided herein arejust a few examples of COP1 sequences that can be readily available foruse in the present invention and thus should not be interpreted in anyway to limit the scope of the present invention. The COP1 nucleotidesequence used in the present invention can be a full length or afragment of any of the COP1 nucleotide sequences from any species. Thoseskilled in the art will be able to identify other COP1 sequences fromdifferent species and alterations that can be made to the COP1 sequencesand method disclosed herein while not departing from the scope of thepresent invention.

Preparation of cDNA Libraries for Isolation of COP1 Gene

Complementary DNA (cDNA) libraries from a plant may be prepared andscreened for COP1 nucleic acids. Using a maize plant as an exampleherein and throughout the detailed descriptions of the preferredembodiments, cDNA libraries from the maize plant may be preparedaccording to standard techniques known to those skilled in the art, forinstance, in Sambrook et al., Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989). Usingconventional methodologies, cDNA libraries can be constructed from themRNA of a given tissue sample or an organ using poly dT primers andreverse transcriptase (Efstratiadis et al., Cell 7:279-288, 1976;Higuchi et al., Proc. Natl. Acad. Sci. (U.S.A.) 73:3146-3150, 1976;Maniatis et al., Cell 8:163, 1976; Land et al., Nucleic Acids Res.9:2251-2266, 1981; Okayama et al., Mol. Cell. Biol. 2:161-170, 1982;Gubler et al., Gene 25:263, 1983). Several methods may be employed toobtain full-length cDNA constructs. For example, terminal transferasecan be used to add homopolymeric tails of dC residues to the free 3′hydroxyl groups (Land, et al., Nucleic Acids Res. 9:2251-2266, 1981).This tail can then be hybridized by a poly dG oligo which can act as aprimer for the synthesis of full length second strand cDNA. A simplifiedmethod has been developed by using synthetic primer-adapters that haveboth homopolymeric tails for priming the synthesis of the first andsecond strands and restriction sites for cloning into plasmids(Coleclough et al., Gene 34:305-314, 1985) and bacteriophage vectors(Krawinkel et al., Nucleic Acids Res. 14:1913, 1986; and Han et al.,Nucleic Acids Res. 15: 6304, 1987).

A method to enrich preparations of mRNA is to fractionate by size. Onesuch method is to fractionate by electrophoresis through an agarose gel(Pennica et al., Nature 301:214-221, 1983). Another such method employssucrose gradient centrifugation in the presence of an agent, such asmethylmercuric hydroxide, that denatures secondary structure in RNA(Schweinfest, et al., Proc. Natl. Acad. Sci. (U.S.A.) 79:4997-5000,1982).

In one of the preferred embodiments, preparation of appropriatelyenriched cDNA libraries from tissue of interest such as a tissue samplefrom the stem or ear of the maize plant may be described as below. Themaize plants may be grown in a greenhouse and, when they reach a desireddevelopmental stage, they may be used for collection of the tissuesamples. The cDNA library may be constructed using techniques known tothose skilled in the art. Briefly, mRNA from the tissue sample may beisolated and cDNA prepared. Short chains of oligo d-T nucleotides may behybridized with the poly-A tails of the mRNA and serve as a primer forthe enzyme, reverse transcriptase, which synthesizes a complementary DNA(cDNA) strand. The cDNA may be enriched for the desired sequences usingsubtraction hybridization procedures following Davis et al. (Proc. Natl.Acad. Sci. USA 81: 2194-2198, 1984). The quality of the cDNA library maybe determined by examining the cDNA insert size, and also by sequenceanalysis of a random selection of an appropriate number of clones fromthe library.

Amplification of the COP1 Gene from the cDNA Library

As described herein, COP1 nucleic acid molecules from the cDNA from themaize plant may be amplified through use of many available methods. Themost preferred method of achieving such a goal may employ the polymerasechain reaction, i.e., “PCR” (Mullis et al., Cold Spring Harbor Symp.Quant. Biol. 51:263-273, 1986; Erlich et al., European PatentApplication 50,424; European Patent Application 84,796, European PatentApplication 258,017, European Patent Application 237,362; Mullis,European Patent Application 201,184; Mullis et al., U.S. Pat. No.4,683,202; Erlich., U.S. Pat. No. 4,582,788; and Saiki et al., U.S. Pat.No. 4,683,194), using primer pairs that are capable of hybridizing tothe proximal sequences that define the COP1 nucleic acid of the cDNAlibrary in its double-stranded form.

The COP1 nucleic acid molecules may also be amplified by alternativemethods, such as the “Ligase Chain Reaction”, i.e., LCR (Barany, Proc.Natl. Acad. Sci, (U.S.A.) 88:189-193, 1991). LCR uses two pairs ofoligonucleotide probes to exponentially amplify a specific target. Thesequences of each pair of oligonucleotides are selected to permit thepair to hybridize to abutting sequences of the same strand of thetarget. Such hybridization forms a substrate for a template-dependentligase. As with PCR, the resulting products thus serve as a template insubsequent cycles and an exponential amplification of the desiredsequence is obtained.

Other known nucleic acid amplification procedures, such asallele-specific oligomers, branched DNA technology, transcription-basedamplification systems, oligonucleotide ligation assay, or isothermalamplification methods may also be used to amplify and analyze the COP1nucleic acid molecules from the cDNA library of a plant such as themaize plant (Malek et al., U.S. Pat. No. 5,130,238; Davey et al.,European Patent Application 329,822; Schuster et al., U.S. Pat. No.5,169,766; Miller et al., PCT Application WO 89/06700; Kwoh et al.,Proc. Natl. Acad. Sci. (U.S.A.) 86:1173-1177, 1989; Landegren et al.,Science 241: 1077-1080, 1988; Gingeras et al., PCT Application WO88/10315; Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396,1992).

Sequencing of the COP1 Nucleic Acid from the cDNA Library

The COP1 nucleic acid molecule of the cDNA library from the maize plantmay be sequenced after its amplification through use of many availablemethods. The most preferred method of achieving such a goal may employthe polymerase chain reaction (“PCR”), as described above, using primerpairs that are capable of hybridizing to the proximal sequences thatdefine the COP1 cDNA library in its double-stranded form.

Antibody Production

In one of the preferred embodiments, antibodies to the maize COP1 of thepresent invention may be produced using standard immunologicaltechniques for production of polyclonal antisera and, if desired,immortalizing the antibody-producing cells of the immunized host forsources of monoclonal antibody production. Techniques for producingantibodies to any substance of interest are well known, e.g., as inHarlow and Lane (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988) and as in Goding (MonoclonalAntibodies: Principles and Practice, 2^(nd) eds, Academic Press, N Y,1986). The antibodies produced in the present invention are useful inimmunoassays for determining the amount or presence of the COP1 protein.Such assays are also useful in quality controlled production ofcompositions containing COP1 of the present invention. In addition, theantibodies can be used to assess the efficacy of recombinant productionof the COP1, as well as for screening expression libraries for thepresence of COP1 encoding gene. They may also be used as affinityligands for purifying and/or isolating the COP1 proteins. The COP1antigens may be obtained by over expressing the full or partial lengthof the COP1 gene.

Promoter Selection and Vector Construction

Exogenous genetic material such as the wild type COP1 nucleic acid orits fragment thereof may be transferred into a plant cell by use of aDNA vector or construct designed for such a purpose. Design of such avector is generally within the skill of the art (See, Plant MolecularBiology: A Laboratory Manual, Clark eds, Springer, New York, 1997).

In one of the preferred embodiments, the construct may be an antisenseconstruct comprising the COP1 nucleic acid that is complementary to, andis capable of pairing, with the native COP1 mRNA and thus preventtranslation of the native COP1 mRNA. See Mol et al. (FEBS Lett. 268:427-430, 1990) and Green et al. (Annu. Rev. Biochem. 55: 569-597, 1986)for general description of the technique. An antisense vector may beconstructed by standard procedures and introduced into cells.

In another preferred embodiment, the construct may be a regulartransformation vector and the process involves a “dominant-negative”approach to reduce the functions of native COP1 proteins. In such amethod, part or all of the COP1 normal nucleic acid sequence is placedunder the control of a promoter so that a partial or whole sequence of aprotein similar to the targeted native protein is produced in small orlarge quantity. These partial or whole sequence of the expressed COP1proteins may interact with the native COP1 proteins in such a way thatthe expression level and function of the native COP1 proteins bereduced. Because of the dominant-negative response of the endogenouscop1 alleles, this process will modify the shade-avoidance response tocause production of dominant-negative transgenic plants.

A construct or vector may include a plant promoter to express a COP1nucleic acid or a fragment thereof. Promoters which are known or foundto cause transcription of nucleic acid molecules can be used for DNAtranscription in the maize plants. Such promoters may be obtained from avariety of sources such as plants and plant viruses. The promoterselected should not cause any potential problems for plant's growth anddevelopment. For example, the promoter selected should not cause anyseed germination problems. A number of promoters which are active inplant cells have been described in the literature and have been used tocreate DNA constructs which have been expressed in plants (see, e.g.,PCT publication WO 84/02913). For the purpose of the present invention,it is preferred that the particular promoter selected should be alight-inducible promoter. This light-inducible promoter should be, inthe case of overexpressing the COP1 binding domains, capable of causingsufficient expression of the exogenous COP1 so that the exogenous COP1proteins that include the protein-binding domains will be produced at ahigher level to cause the binding activities in the transformants toresult in the inactivity of the indigenous COP1 proteins. Thislight-inducible promoter should be, in the case of using the antisensetechnology, capable of producing an effective amount of mRNA from theexogenous COP1. Thus the effective amount of mRNA so produced will bindto the indigenous mRNA in the “antisense” orientation and causesuppression of the COP1. In either of the above two events, since theindigenous COP1 expression is suppressed, the desired phenotype will inexpectation have shorter internodes. The promoters suitable for thepresent invention may include a cab promoter, an ATHB-2 promoter, a riceHB-2 promoter and a corn HB-2 promoter. In one of the preferredembodiments, the promoter may be the cab promoter. The methods foridentifying and isolating a light-inducible promoter for the presentinvention can be readily available (e.g., Sheen, Plant Cell 2:1027-1038, 1990).

In addition to promoters which are known to cause transcription of COP1in plants as described above, other promoters may be identified for usein the present invention by screening a plant cDNA library for nucleicacids which are selectively or preferably expressed in the targettissues or cells.

The vector or construct may also include a structural gene or a fragmentof the structural gene thereof. The “structural gene” or a fragment ofthe “structural gene” as used herein in the present invention comprisesthe COP1 gene or a fragment of the COP1 gene. The COP1 gene may beoperatively linked downstream to a promoter as described above. In oneof the preferred embodiments, the COP1 gene may be a wild type COP1nucleic acid or a portion of the COP1 nucleic acid from any source. TheCOP1 nucleic acid may be from a maize plant and may be the ZmCOP1nucleic acid as disclosed in the present invention having SEQ ID NO: 12.

The vector or construct may also include, within the coding region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. For example, such sequences thathave been isolated include the Tr7 3′ sequence and the nos 3′ sequence(Ingelbrecht et al., The Plant Cell 1:671-680, 1989; Bevan et al.,Nucleic Acids Res. 11:369-385, 1983) or the like.

The vector or construct may also include regulatory elements. Examplesof such regulatory elements may include the Adh intron 1 (Callis et al.,Genes and Develop. 1:1183-1200, 1987), the sucrose synthase intron(Vasil et al., Plant Physiol. 91:1575-1579, 1989), the TMV omega element(Gallie et al., The Plant Cell 1:301-311, 1989), and maize heat shockprotein 70 (hsp70) intron (Brown and Santino, PCT ApplicationWO93/19189). These and other regulatory elements may be included whenappropriate.

The vector or construct may also include a selectable marker, ascreenable marker and other elements as appropriate. Examples of theseelements and markers mentioned herein are known in the art and may bereadily used in the present invention. Those of the skilled in the artshould refer to the following art for details (for selectable markers,see Potrykus et al., Mol. Gen. Genet. 199:183-188, 1985; Hinchee et al.,Bio. Technology 6:915-922, 1988; Stalker et al., J. Biol. Chem.263:6310-6314, 1988; European Patent Application 154,204; Thillet etal., J. Biol. Chem. 263:12500-12508, 1988; for screenable markers see,Jefferson, Plant Mol. Biol. Rep. 5: 387-405, 1987; Jefferson et al.,EMBO J. 6: 3901-3907, 1987; Sutcliffe et al., Proc. Natl. Acad. Sci.(U.S.A.) 75: 3737-3741, 1978; Ow et al., Science 234: 856-859, 1986;Ikatu et al., Bio. Technol. 8: 241-242, 1990; and for other elementssee, European Patent Application Publication Number 0218571; Koziel etal., Plant Mol. Biol. 32: 393-405; 1996).

Methods and compositions for transforming bacteria and othermicroorganisms are known in the art (see, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Plant Transformation

The COP1 nucleic acid molecules of the present invention may betransferred into a plant cell and the plant cell regenerated into awhole plant. The COP1 nucleic acid molecules may be from any source,whether naturally occurring or otherwise obtained through methodologiesin the field that are readily known to those skilled in the art, thatare capable of being inserted into any plant cells. The COP1 nucleicacid molecules may be transferred into either monocotyledonous anddicotyledonous plants (See specifically, Chistou, Particle Bombardmentfor Genetic Engineering of Plants, Pp. 63-69 (Zea mays), BiotechnologyIntelligence Unit. Academic Press, San Diego, Calif., 1996), andgenerally Chistou, Particle Bombardment for Genetic Engineering ofPlants, Biotechnology Intelligence Unit. Academic Press, San Diego,Calif., 1996). As an example, the COP1 gene may be transformed into themaize plant using any of the methods as described herein.

There are many methods for transforming the COP1 nucleic acid moleculesinto plant cells such as the maize plant cells. Suitable methods arebelieved to include virtually any methods by which nucleic acidmolecules may be introduced into the cells, such as by Agrobacteriuminfection or direct delivery of nucleic acid molecules that may includePEG-mediated transformation, electroporation and acceleration of DNAcoated particles, etc. (Potrykus, Ann. Rev. Plant Physiol. Plant Mol.Biol. 42:205-225, 1991; Vasil, Plant Mol. Biol. 25: 925-937, 1994). Forexample, electroporation has been used to transform Zea mays protoplasts(Fromm et al., Nature 312:791-793, 1986). In general, the following arefour most commonly used general methods for delivering a gene intocells: (1) chemical methods (Graham and van der Eb, Virology,54:536-539, 1973); (2) physical methods such as microinjection(Capecchi, Cell 22:479-488, 1980), electroporation (Wong and Neumann,Biochem. Biophys. Res. Commun. 107:584-587, 1982); Fromm et al., Proc.Natl. Acad. Sci. (USA) 82:5824-5828, 1985); U.S. Pat. No. 5,384,253; andthe gene gun (Johnston and Tang, Methods Cell Biol. 43:353-365, 1994);(3) viral vectors (Clapp, Clin. Perinatol. 20:155-168, 1993; Lu et al.,J. Exp. Med. 178:2089-2096, 1993; Eglitis and Anderson, Biotechniques6:608-614, 1988); and (4) receptor-mediated mechanisms (Curiel et al.,Hum. Gen. Ther. 3: 147-154, 1992; Wagner et al., Proc. Natl. Acad. Sci.(USA) 89: 6099-6103, 1992).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. See for example(Potrykus et al., Mol. Gen. Genet., 205:193-200, 1986; Lorz et al., Mol.Gen. Genet., 199:178, 1985; Fromm et al., Nature, 319:791, 1986;Uchimiya et al., Mol. Gen. Genet.: 204:204, 1986; Callis et al., Genesand Development, 1183, 1987; Marcotte et al., Nature, 335:454, 1988).Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Among them are the methods for corn (U.S. Pat. Nos. 5,569,834,5,416,011; McCabe et al., Biotechnology 6:923, 1988; Christou et al.,Plant Physiol., 87:671-674, 1988). Illustrative methods for theregeneration of cereals from protoplasts are also described (Fujimura etal., Plant Tissue Culture Letters, 2:74, 1985; Toriyama et al., Theor.Appl. Genet. 205:34, 1986; Yamada et al., Plant Cell Rep. 4: 85, 1986;Abdullah et al., Biotechnology, 4:1087, 1986).

In one of the preferred embodiments, the present invention employs theAgrobacterium-mediated transformation technology to introduce the COP1nucleic acid into the maize plant and to achieve a desired result.Agrobacterium-mediated transfer is a widely applicable system forintroducing genes such as the COP1 gene into plant cells because thegene such as the COP1 gene can be introduced into whole plant tissues,thereby bypassing the need for regeneration of an intact plant from aprotoplast. The use of Agrobacterium-mediated plant integrating vectorsto introduce a nucleic acid into plant cells is well known in the art.See, for example, Fraley et al. (Biotechnology 3:629-635, 1985), Hiei etal. (U.S. Pat. No. 5,591,616), and Rogers et al. (Meth. In Enzymol 153:253-277, 1987), Further, the integration of the Ti-DNA is a relativelyprecise process resulting in few rearrangements. The region of the COP1nucleic acid to be transferred is defined by the border sequences and isusually inserted into the plant genome as described in Spielmann et al.(Mol. Gen. Genet., 205:34, 1986).

A transgenic plant such as a transgenic maize plant formed usingAgrobacterium transformation methods typically contains a single addedCOP1 gene on one chromosome. Such a transgenic plant can be referred toas being heterozygous for the added COP1 gene. More preferred is atransgenic plant that is homozygous for the added COP1 gene; i.e., atransgenic plant that contains two added COP1 genes, one gene at thesame locus on each chromosome of a chromosome pair. A homozygoustransgenic plant can be obtained by sexually mating (selfing) anindependent segregated transgenic plant that contains a single addedCOP1 gene, germinating some of the seeds produced and analyzing theresulting plants produced for the COP1 gene.

It is understood that two different transgenic plants can also be matedto produce offspring that contain two independently segregating added,exogenous COP1 genes. Selfing of appropriate progeny can produce plantsthat are homozygous for both added, exogenous COP1 genes that encode aCOP1 polypeptide. Back-crossing to a parental plant and out-crossingwith a non-transgenic plant are also contemplated, as is vegetativepropagation.

Regeneration of the Transformed Plants

The regeneration, development, and cultivation of plants such as themaize plants from transformants or from various transformed explants arewell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, Eds., Academic Press, Inc. San Diego, Calif., 1988).This regeneration and growth process may typically include the steps ofselection of transformed cells containing exogenous COP1 genes,culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

The regeneration of plants containing the foreign, exogenous gene thatencodes a protein of interest is well known in the art. As described inthe present invention, the regenerated plants such as the regeneratedmaize plants that contain the COP1 nucleic acids, either wild type orchemically synthesized, that encode for the COP1 proteins, may bepreferably self-pollinated to provide homozygous transgenic maizeplants, as discussed before. Otherwise, pollen obtained from theregenerated maize plants may be crossed to seed-grown plants ofagronomically important lines. Conversely, pollen from plants of theseimportant lines is used to pollinate regenerated plants. A transgenicmaize plant of the present invention may be cultivated using methodswell known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. Monocotyledonous plants, or monocot plants, may betransformed with a COP1 nucleic acid and then regenerated.Transformation of monocot plants using electroporation, particlebombardment, and Agrobacterium has also been reported. Transformationand plant regeneration have been achieved in many monocot plants thatinclude maize, asparagus, barley and wheat, etc. Dicotyledonous plants,or dicot plants, may also be transformed with COP1 nucleic acid andregenerated. Methods for transforming dicot plants, primarily by use ofAgrobacterium tumefaciens, and obtaining transgenic plants have beenpublished. Among them are the methods for soybean, cotton, and otherdicot plants.

Monocot and dicot plants to which the present invention may be appliedmay include those agronomic and horticultural crop plants. Examples ofagronomic crop plants may include cereals such as maize, wheat, rye,barley, oats, buckwheat, sorghum and rice; non-cereals such assunflower, canola, peas, beans, soybeans, cotton and linseed; vegetablessuch cauliflower, asparagus, lettuce, tobacco and mustard; and rootcrops such as sugarbeet, potato, sweet potato, carrot and turnip.Horticultural crops may include celery, tomato, egg plant, cucumber andsquash. Fruit crops may include apple, apricot, peach, pear, plum,orange, blackberry, blueberry, strawberry, cranberry and lemon.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, 1989; Mailga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor,N.Y., 1997).

The following examples further demonstrate several preferred embodimentsof the present invention. Those skilled in the art will recognizenumerous equivalents to the specific embodiments described herein. Suchequivalents are intended to be within the scope of the present inventionand claims.

EXAMPLES Example 1. Computer-Aided Sequence Analysis

A COP1 protein sequence from Arabidopsis was retrieved from GenBank (ID402685) and used as query to BLAST search sequence databases whichidentified clones in the databases that share high degree homology toArabidopsis COP1. These clones may be used in cloning the full-lengthCOP1 cDNA from these crop species from which the clones were originallyobtained including maize.

Example 2. COP1 cDNA Cloning

To obtain the full-length sequence of maize COP1 cDNA, several RT-PCR,5′race and 3′race polymerase chain reactions (PCR) were performed. Onemicrogram of kernel cDNA, recovered from kernels 15 days afterpollination (DAP) was used as template in all PCR reactions. The cDNAlibrary had previously been cloned into pSPORT2. Two micromoles of maizeCOP1 gene specific forward primer L3062COP IF(5′GTACGGACATTCAGAGGACAC3′; SEQ ID NO: 1) and reverse primerL30623′COP1R (5′GTGTCCTCTGAATGTCCGTAC3′; SEQ ID NO: 2) combined with 0.1mM dNTPs and 5 Units of Taq DNA polymerase was used in a 50 μL PCRreaction to determine the presence of COP1 in the 15 DAP kernel cDNApools. PCR cycling conditions and parameters were as follows: 95° C. for5 minutes (min) followed by 28 cycles at 95° C. for 30 seconds (sec),60° C. for 30 sec and 72° C. for 30 sec. To obtain the most 5′ sequencesof COP1 cDNA, the complementary sequences of M13 Forward primer(5′CCCAGTCACGACGTTGTAAAACG3′; SEQ ID NO: 3) in pSPORT2 vector and theprimer L3062cop1R, combined with 0.1 mM dNTPs and 5 units of HotStartTaq™ polymerase (Qiagen, Valencia, Calif.), were used in a 50 μL in a5′-race PCR. PCR cycling conditions and parameters were as follows: 95°C. for 15 min, then 28 cycles at 95° C. for 30 sec, 60° C. for 30 secand 72° C. for 3 min. An approximately 2 Kb PCR product was obtained,cloned into PCR-blunt TOPO II cloning vector (Invitrogen, Carlsbad,Calif.) and sequenced. In the meantime, COP1 gene specific primerL30624070F (5′AATGAAAAGAACTTTGTTGGC3′; SEQ ID NO: 4) and M13 reverseprimer (5′AGCGGATAACAATTTCACACAGG3′; SEQ ID NO: 5) were mixed with 0.1mM dNTPs and 5 Units Taq DNA polymerasein a 50 μL 3′race PCR to obtainthe most 3′sequences of the COP1 cDNA. PCR cycling conditions andparameters were as follows: 95° C. for 5 min, then 28 cycles at 95° C.for 30 sec, 60° C. for 30 sec and 72° C. for 40 seconds. A 650 base pairPCR product was obtained, cloned into PCR-blunt TOPO II cloning vector,and sequenced. For subcloning purposes, and for obtaining a morereliable cDNA clone, COP1 cDNA was re-amplified from 15 DAP kernel cDNAlibrary using forward primer COP15-6 (5′CTGCGCCATGGGCGACTCCTCGGTGG3′;SEQ ID NO: 6) containing NcoI site at the start codon of the COP1 openreading frame and reverse primer L30623′COP1R (SEQ ID NO: 2). A 50 μL ofPCR cocktail was made as follows: 1 μg of 15 DAP kernel cDNA, 2 μM ofCop15-6 and L30623′Cop1R primers, 0.1 mM dNTPs, 5% DMSO, and 1×PCRreaction buffer (Mg++). Manual hot start PCR cyclings were initiated at95° C. for 15 min. Then 2 units of Expand™ High-Fidelity DNA Polymerase(Roche, 173264) was added and PCR reaction was carried out under thefollowing conditions and parameters: at 95° C. for 30 seconds, 60° C.for 30 sec and 68° C. for 6 min for a total of 28 cycles. A 2230 basepair maize COP1 cDNA sequence was obtained, and cloned into PCR-BluntTOPO II cloning vector. The full-length COP1 cDNA sequence was confirmedby sequencing six independent clones. The sequence was named as ZmCOP1(SEQ ID NO: 12). ZmCOP1 represents a full length cDNA sequencecontaining a 388 base pair 5′ UTR, a coding region that encodes 694amino acids (see translated amino acid sequence; SEQ ID NO: 13) and a141 base pair 3′ UTR. Based upon sequence homology analysis, theisolated maize COP1 nucleotide sequence has 50% and 71% identity withthose of COP1 from Arabidopsis (g17446130, Deng et al., Cell 27,791-801, 1992) and rice (gi7592844), respectively (FIGS. 1 and 2). Themaize COP1 protein sequence is also aligned with other COP1 proteinsequences from other species and shows 68% sequence identity to that ofpea (gi3121867, Zhao et al., Biochimica et Biophysica Acta-GeneStructure and Expression 1395, 326-328, 1998), 69% to Japanese morningglory (gi11127996), 70% to Arabidopsis (gi7446130, Deng et al., Cell 27,791-801, 1992), 71% to tomato (gi4090943), and 89% to rice (g17592844),respectively (FIG. 3). The identity levels were determined by BLASTprogram (Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997) withdefault parameters (Expectation value (E): blank; Alignment viewoptions: pairwise; Filter query sequence: no; Cost to open a gap: 0;Cost to extend a gap: 0; X dropoff value for gapped alignment: 0; ShowGI's in defines: no; Penalty for a nucleotide mismatch: −3; Reward for anucleotide match: 1; Threshold for extending hits: 0; Perform gappedalignment: yes; Query Genetic code to use: standard; DB Genetic code:standard; Believe the query define: no; Matrix: BLOSUM62; Word size: 0;Effective length of the database: 0; Query strand Use: both).

Example 3. Northern Blot Analysis of COP1 from the cDNA Library

Northern Blot analysis of the expression of maize COP′ during eardevelopment was performed. Total RNA (15 μg each) from different tissueswere mixed with one volume of RNA Sample Loading Buffer (Sigma, R-4268),heated at 65° C. for 10 min., chilled on ice for one min. and loaded ona 1% formaldehyde agarose gel. The RNA was separated on the gel underconstant voltage at 65 v for 4 to 5 hours and then transferred onto apiece of nylon membrane at 4° C. overnight using a Schleicher & Schuelltransfer system (Keene, N.H., USA 03431). A 157 base pair 3′UTR fragmentof COP1 was amplified by PCR using forward primer 3′COP1(5′TGCTCCTTGATGTTATGG3′; SEQ ID NO: 7) and reverse primer L30623′COP1R.A 910 base pair 5′ fragment was also amplified by PCR using forwardprimer COP15-6 and COP301 (5′GATGAATTCATCAAGGAGGATCAGAAGAAG3′; SEQ IDNO:8). Purified PCR products (25 ng each) were labeled with p32dCTPusing Random Primed DNA Labeling Kit (Boehringer Mannheim, Cat.#1004760). The membrane was hybridized with both labeled 3′UTR and5′COP1 probes.

Example 4. Construction of Maize Transformation Vectors

In an effort to identify an efficient way for reducing the endogenousCOP1 mRNA and protein level, three sequences of the COP1 gene withdifferent lengths were created and dominant negative and antisensestrategies were employed. The dominant negative strategy employed afragment of the 5′ nucleotide sequence encoding a protein binding domainwas cloned into an overexpression vector. Two sequences employing COP1were created for this purpose, one containing the first 1233 nucleotideresidues (SEQ ID NO: 14) encoding 411 amino acids (SEQ ID NO: 15) inpMON47119 (FIG. 4), and another containing the first 906 nucleotideresidues (SEQ ID NO: 16) encoding 301 amino acids (SEQ ID NO: 17) inpMON47118 (FIG. 5). A full length COP1 was used in the antisenseapproach. The construct was pMON47120 and the detail of the vector wasshown in FIG. 6. Forward primer of COPantisense5′-end(5′GATGAATTCCTGCGGCATGGGCGAC3′; SEQ ID NO: 9) and reverse primer ofCOPantisense3′-end (5′AACCATGGACTGAACCTCTTGAACG3′; SEQ ID NO: 10) wereused to sub-clone full-length maize COP1 for the antisense strategy.Primers COP15-6 and COP301 were used to sub-clone N-terminal 301 (N301)amino acid protein domain of maize COP1. This maize N301 protein domainwas equivalent to N 282 protein dimerization domain of Arabidopsis COP1(Deng et al., PCT Application WO 00/18940). Forward primer of COP15-6and reverse primer of COP411 (5′GATGAATTCATCATTTCGAGACTCCAGC3′; SEQ IDNO: 11) with two stop codons (TGA) near EcoRI cleavage site were used tosub-clone the N-terminal 411 (N411) amino acid protein domain of themaize COP1. This N411 fragment was equivalent to the N392 fragment COP1in Arabidopsis that contained the protein dimerization domain and coresequences required for COP1 protein translocation from cytoplasm tonucleus (PCT Application WO 00/18940). PCR mixture (50 μL) was made asfollows: 100 ng of PCR-Blune-COP1 plasmid DNA, 2 μM of primers, 0.1 mMdNTPs, 5% DMSO, 1×PCR buffer, and 5 U Expand High Fidelity DNApolymerase. PCR cycling conditions and parameters were as follows: 95°C., 5 min., followed by 25 cycles at 95° C. for 30 seconds, 60° C. for30 seconds, 68° C., 6 min. for antisense COP1 amplification, 2 min. forCOPaa301 amplification, and 3 min for COPaa411 amplification. The PCRproducts were gel purified, cloned into PCR-Blunt TOPO II cloningvector, and full-length sequenced.

To construct maize Agrobacterium-mediated transformation vectors,pMON32502 plasmid DNA was digested with HindIII, EcoRI and NcoI. A 3135base pair HindIII and EcoRI vector backbone fragment was isolated.Plasmid DNA of pMON24037 was digested with HindIII and NcoI. A 1689 basepair of the HindIII and NcoI fragment containing the promoter of maizechlorophyll alb binding protein and the hsp70 intron was obtained.PCR-Blunt vector containing antisense COP1, COP1aa301, and COP1aa411were digested respectively with NcoI and EcoRI to obtain the NcoI andEcoRI fragments of antisense COP1, COP1aa301 and COP1aa411. The HindIIIand EcoRI vector backbone fragment was ligated with the HindIII and NcoIcab promoter fragment, and NcoI and EcoRI antisense COP1, or COPaa301,or COPaa411, respectively, to form pMON47120, pMON47118, and pMON47119.The three constructs were then transformed individually intoAgrobacterium strain ABI. All these gene constructs were under thecontrol of cab promoter (light inducible) and were designed to reducethe functional ZmCOP1 protein level in the above-ground part of cornplants. The vector pMON47118 contained the coding sequence forN-terminal 301 amino acid residues of the ZmCOP1 gene that covered thedimmerization domain. The vector pMON47119 contained the nucleotidesequence coding for the 411 amino acid residues. The N-terminus ofZmCOP1 carried the dimerization and nuclear localization domains.pMON47120 contained the full-length antisense ZmCOP1 gene.

To serve as a check for evaluation of ZmCOP1 functions in corn, twoadditional ZmCOP1 constructs, i.e., pMON47130 and pMON47131, were madefor transformation. A fragment of ZmCOP1 gene containing 903 nucleotideresidues was driven by a constitutive promoter, i.e., a rice-actin(RACT) promoter. In order to construct pMON47130 transformation vector,a plasmid DNA of pMON47123 was digested with NcoI and XhoI restrictionenzymes. A 1404 base-pair XhoI and NcoI fragment which containedrice-actin promoter (RACT) was obtained from pMON47123. Plasmid DNA ofpMON47118 was also digested with NcoI and XhoI restriction enzymes. Afragment containing 8458 bp from 4379 bp XhoI site to 2719 bp NcoI sitewas obtained from pMON47118. Then, the 1404 bp fragment was ligated tothe 8458 bp fragment and pMON47130 construct was made (FIG. 7). In orderto make pMON47131 transformation vector, a plasmid DNA of pMON47120 wasdigested with NcoI and XhoI restriction enzymes. A fragment containing9567 bp from 5594 bp XhoI site to 3934 bp Neal site on pMON47120 wasobtained. The RACT promoter fragment obtained from pMON47123 asdescribed above was ligated to the 9567 bp fragment and the construct,i.e., pMON47131, was made (FIG. 8). The vector contained the full lengthZmCOP1 coding sequence in reverse orientation.

Example 5. Overexpression of ZmCOP1 N301 Domain for Antibody Production

In order to make polyclonal antibody for maize COP1 protein, plasmid DNAof pMON47118 and pET30(a) (Novagen, Madison, Wis.) were digested withNcoI and EcoRI The NcoI and EcoRI COPaa301 fragment was directionallycloned into pET30(a) vector under the control of the IPTG inducible T7polymerase promoter and was transformed into E. coli BL21(DE3) competentcells. E. coli BL21 (DE3) cells containing pET30(a)-COPaa301 constructwere induced with 3 mM IPTG overnight at room temperature. Proteins werepurified under denaturing conditions using Ni-NTA Superflow resin(QIAGEN, Valencia, Calif.) as described in the manufacturer's protocol.An about 38 kDa His-tag COP301 protein was purified and confirmed byWestern Blot analysis using monoclonal antibody against S-tag protein.About 1 mg purified COPaa301 protein was used to inoculate two rabbitsfor antibody production following the standard protocol of Pocono RabbitFarm & Laboratory Inc. (Canadesis, Pa.). The preimmune serum from thesetwo rabbits showed no reactivity with COPaa301 protein.

Example 6. Transformation of Corn Plant with the Vectors 1) PlantMaterials

Ears from LH172 were obtained mostly from greenhouses and were usuallyharvested about 10 to 11 days post pollination. Before isolation, earswere stored from 0 to 5 days at 4° C. Ears were sterilized for 20 min in50% (v/v) commercial bleach (Clorox®, with 5.25% sodium hypochlorite)followed by 3 rinses with sterile water.

TABLE 2 Media used in corn transformation (per liter). Co-cultureComponent ½ MS VI ½ MS PL medium LH172 MS MS/BAP MSOD MS salts .1 g 0.1g 2.2 g 4.4 g 4.4 g .1 g Sucrose 20 g 0.1 g 20 g 30 g 30 g — Maltose — —— — — 40 g Glucose 10 g 36 g 10 g — — 20 g 1-Proline 0.115 g 0.115 g0.115 1.36 g 1.36 g — Casamino Acids — — — 0.05 g 0.05 g — Glycine 2 mg2 mg 2 mg — — — 1-Asparagine — — — — — 150 mg myo-Inositol 100 mg 100 mg100 mg — — 100 mg Nicotinic Acid 0.5 mg 0.5 mg 0.5 mg 0.65 mg 0.65 mg0.65 mg Pyridoxine-HCl 0.5 mg 0.5 mg 0.5 mg 0.125 mg 0.125 mg 0.125 mgThiamine-HCl 0.1 mg 0.1 mg 0.6 mg 0.125 mg 0.125 mg 0.125 mg CaPantothenate — — — 0.125 mg 0.125 mg 0.125 mg 2,4-D — — 3 mg 0.5 mg 0.5mg — Picloram — — — 2.2 mg .1 mg — Silver Nitrate — — — 3.4 mg — —Na-Thiosulfate — — — — — — Phytagar — — — 7.0 g 7.0 g 7.0 g Low EEOagarose — — 5.5 g — — —

2) Agrobacterium Induction and Inoculation

Agrobacterium tumefaciens (ABI strain) was grown in LB liquid medium (50mL medium per 250 mL flask) containing 100 mg/L kanamycin and 50 mg/Lspectinomycin for an initial overnight propagation (on a rotary shakerat 150 to 160 rpm) at 27° C. Ten mL of the overnight Agrobacteriumsuspension was transferred to 50 mL of fresh LB in a 250 mL flask (samemedium additives and culture conditions as stated above) and grown forapproximately 8 hours. Suspension was centrifuged around 3500 rpm andpellet resuspended in AB minimal medium (now containing ½ the level ofspectinomycin and kanamycin used for LB) containing 100 μMacetosyringone (AS) for a final concentration of 0.2×10⁹ cfu/mL (or anOD of 0.2 at 660 nm). These Agrobacterium cultures were allowed toincubate as described above for approximately 15 to 16 hours. TheAgrobacterium suspension was harvested via centrifugation and washed in½ MS VI medium (Table 2) containing AS. The suspension was thencentrifuged again before being brought up in the appropriate amount of ½MS PL (Table 2) (also containing AS) so that the final concentration ofAgrobacterium was 1×10⁹ cfu/mL (which is equal to an OD of 1.0 at 660run). Immature embryos from each ear of a LH172 plant (1.5 mm to 2.0 mmlong) were directly dissected into a 1.5-mL eppendorf tube with ½ MS PLcontaining Agrobacterium at an OD of 1.0. The eppendorf tube was cappedtightly and inverted 3 times so that embryos inside were fully immersed.About half of the solution in the tube was drained by using a sterileSAMCO transfer pipette. The rest of the solution together with theembryos were poured into 2-3 layers of sterile Baxter filter paper (5.5cm in diameter). Embryos were removed from the filter paper by flippingthe filter paper over and slightly pressing it against the co-culturemedium (Table 2) with the addition of 20 μM silver thiosulfate in thepetri dish. The embryos were cultured at 23° C. for 1 day and then weretransferred to the first selection medium (LH172MS; Table 2).

3) Callus Induction and Selection (in Dark)

Selection was performed in LH172 medium with 500 mg/L carbenicillin and100 mg/L paromomycin. Three transfers to new plates containing the samemedium were made every two weeks.

4) Regeneration (in Light)

Paromomycin resistant callus was first moved to MS/BAP medium (Table 2)with with 3.5 mg/L 6-BA for 5 to 7 days. After the 6-BA pulse, calluswith green shoot tips were moved to MSOD (Table 2) with 100 mg/Lparomomycin plates and were cultured for another 10 to 12 days. Afterthis stage, green shoots started to grow out as well as white roots.Those small plantlets were transferred to phytatray (1 event perphytatray) containing MSOD media with 100 mg/L paromomycin. After 2 to 3weeks, plants were ready to be transplanted into soil. Plants wereacclimated in the growth chamber for week and then moved to thegreenhouse for hardening. Plants were screened for the presence of nptIIafter 3 to 5 days of the hardening process. Only nptII positive plantswere considered for further experimentation.

Example 7. RD and R1 Transgenic Plants

A total of 30 events from pMON47118 construct were selected from 137 R0plants (79 events) based on the expression levels of gene of interest(GOI) by Northern and Western Blot analyses. Among the 30 events, 8events had only F1 seeds and 22 events had R1 seeds. Twenty R1 or F1seeds of each of these events were planted in a greenhouse. Phenotypeobservations were conducted weekly including germination, leaf color,plant height, growth rate, tassel morphology, ear morphology and earnumber. The average height and growth rate of several events were lowercompared to the wild type plants growing nearby (FIG. 9). In comparisonof wild type and transformed adult plants, several transformed R1 plantsin the event 535307 showed better ear growth, i.e., more and larger earsat third and fourth nodes, and distinct ear morphology, i.e., longerhusk leaves. These phenotypic changes may be attributed to the COP1transgene. Variations among individual plants in an event will beexamined for correlation to the presence and expression level of theCOP1 transgene, Cells transformed with a full length or a fragment ofZmCOP1 is used to produce young corn plants using standard protocols.These plants are called R0 generation plants. R0 plants are generatedfrom many transformation events. These plants are grown in greenhouseand screened for the presence of ZmCOP1 transgene by PCR. The messengerRNA and protein expression level of the transgene in R0 plants areexamined by Northern and Western blotting techniques. Events areselected based on the presence and expression level of the transgene inthe R0 plants. The selected events are planted as R1 plants. R1 plantsare examined for the presence of the transgene, the transgene expressionlevel and the expected phenotypic traits such as short internodes andbetter ear development. All the data are used in selecting R1 plants forR2 evaluation. Those R1 plants that show a moderate to high level oftransgene expression and a desired phenotype (shorter internodes andbetter ear development at high density) are chosen. R2 plants areplanted in field as pedigree lines. The zygosity of each line isdetermined by the presence of the transgene in each plant in the lineand by the positive/negative segregation ratio of its R1 predecessor. Afew lines are selected based on their phenotype, transgene expressionand homozygosity. These lines are crossed with another one or moreinbred lines to make hybrid seeds. The hybrid seeds are planted atdifferent density (20,000, 30,000 and 40,000 plants/acre) side by sidewith a control hybrid that is a best yield performer. The lines thatgive the best hybrid yield and the best density regimes are selected forfurther yield testing. The best line or lines proven in these yieldtrails are bulked up and grown in a large scale. For example, grow thenew hybrid at 30,000 plants/acre, resulting in a 10% increase inbiomass. Because the ZmCOP1 transgene is able to reduce shade avoidanceresponse and hold harvest index the same, this results in a 10% yieldgain. In some selected lines, reduced shade avoidance response alsoenhances harvest index; this increases yield even more.

Example 8. Constitutive Expression of ZmCOP1 in Corn

A construct harboring a rice actin promoter and ZmCOP1aa301 genefragment (pMON47130, FIG. 7) was transformed into corn and the eventsnamed YAA. Western blot analysis on the R0 plants showed that some YAAevents have quite high expression of the ZmCOP1aa301 protein (FIG. 10).

F1 seeds from eight events were selected for a dark and a lightexperiment. The dark experiment was conducted in a growth chamber withoptimal conditions for corn seedling growth except that no light wasprovided. At day 5 after shoot emerging from soil, mesocotyl lengths ofeach seedling were measured. Positive and negative segregants wereidentified by Western blotting. Mesocotyl length data did not show asignificant difference between positive and negative segregants. Thisagrees with the results of a similar experiment Arabidopsis. Eightevents of YAA plants were also grown in a growth chamber with 500mmol·m⁻²·s⁻¹ white lights for 14 hours, and dark for 10 hours daily.Positive and negative plants were identified by Western blot analysis ofZmCOP1aa301 protein. Plant heights were measured weekly. The comparisonsof positive and negative plant height are shown in FIG. 2. The resultsindicated that expression of ZmCOP1aa301 in maize using rice actin(RACT) promoter may have resulted shorter plants; while 7 out of 8 eventshowed a trend of shorter stature in the positive plants at V11 stage,the difference in two of these events was statistically significant atp=0.05 and p=0.01 level this sample size and in this growth conditions(FIG. 11).

Shorter Stature in Kyle R3 Plants

Kyle events are the transformants from construct pMON47118, whichcontains a light inducible CAB promoter and ZmCOP1aa301 gene fragment.Western analysis has shown that the ZmCOP1aa301 fragment was expressedin the Kyle plants under normal light (FIGS. 12 and 13) but not in darkor weak light (FIG. 12). Also, the gene was expressed at high level atdifferent growth and developmental stages.

Seeds from some Kyle events were germinated and seedlings grown in darkand under 1 mmol/m2·s dim light and under normal green house light.Mesocotyl and coleoptile length were measured on day 10. Leaf sampleswere taken from each plant for DNA amplification (PCR) of the gene ofinterest. Seedlings grown under full green house light had virtually nomesocotyl, and their coleoptile length is short and uniform among Kylepositive, negative and wildtype plants. When seedlings were grown under1 mmol/m2·s light, their coleoptile and mesocotyl were much longer, butno statistically significant difference was detected between positiveand negative seedlings (FIG. 5).

A greenhouse experiment using R3 homozygous lines of Kyle events wascarried out. Five homozygous positive lines and 5 of their correspondingnegative lines were chosen and 100 seeds of each were planted in theJerseyville greenhouse. PCR results on 12 plants of each line were usedto confirm homozygosity. Thirty-six plants were transplanted for eachline and the positions in the rows were randomized. Plant height data isbeing recorded on a weekly basis since transplanting. Positive plantswere shorter compared to their corresponding negative lines in allevents tested up to VT stage. The difference is statisticallysignificant (Table 1). However, only event Kyle50 maintained thisdifference at maturity.

TABLE 3 Plant height and t-test of 5 Kyle events Height (cm) P-value ofcomparison Event POS NEG LH172 to NEG to LH172 Kyle15 55.19 71.22* 85.83 3.7 × 10⁻⁰⁹* 6.5 × 10⁻¹² Kyle15 63.22 71.22* 85.83  8.1 × 10⁻⁰⁵* 5.5 ×10⁻¹² Kyle17 48.13 69.73 85.83 1.8 × 10⁻⁰⁹ 1.9 × 10⁻¹⁵ Kyle50 36.2465.09 85.83 1.1 × 10⁻¹³ 1.4 × 10⁻¹⁷ Kyle72 63.08 74.21 85.83 2.1 × 10⁻⁰⁵7.4 × 10⁻¹² Kyle77 64.78 79.78 85.83 3.3 × 10⁻⁰⁹ 2.6 × 10⁻¹¹ *Comparedto the average of all negative events

Plant Height of Kurt R1

Kurt events are the transformants from construct pMON47119, whichcontains a light inducible CAB promoter and ZmCOP1aa411 gene fragment.Western analysis of some Kurt events has shown that the ZmCOP1aa411fragment was expressed at high level in many of the events tested (FIG.8). Some R1 and F1 Kurt events were grown in the field. Positive plantswere identified by PCR. The height of both positive and negative plantswas measured on a weekly basis up to VT stage. FIG. 8 summarizes dataobtained at V10 stage, showing that the positive plants were shorterthan their negatives in many events.

In summary, the above describes the present invention. It will beunderstood by those skilled in the art that, without departing from thescope and spirit of the present invention and without undueexperimentation, the present invention can be performed within a widerange of equivalent parameters. While the present invention has beendescribed in connection with specific embodiments thereof, it will beunderstood that it is capable of further modifications. The presentinvention covers any uses, variations, or adaptations of the inventionfollowing the principles of the invention in general. Variouspermutations and combination of the elements provided in all the claimsthat follow are possible and fall within the scope of this invention.

All publications and patents mentioned in this specification are hereinincorporated by reference as if each individual publication or patentwas specially and individually stated to be incorporated by reference.

1. A recombinant DNA construct comprising a COP1 nucleotide sequenceencoding a polypeptide having an amino acid sequence that has at least90% sequence identity to the amino acid sequence of SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or a functionallyequivalent fragment thereof; or wherein said COP1 nucleotide sequencehas at least 90% sequence identity to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:19, SEQ ID NO:20, and afunctionally equivalent fragment thereof; said COP1 nucleotide sequencebeing operably linked to a heterologous promoter, wherein upon itstransformation into a plant said construct causes reduction offunctional endogenous COP1 protein level.
 2. A method for increasingyield of a crop, comprising the steps of: transforming a cell of a cropplant with a nucleic acid molecule comprising a heterologous promoterthat functions in the cells of said crop plant, said promoter operablylinked to a structural nucleic acid sequence, wherein the structuralnucleic acid sequence encodes a constitutive photomorphogenesis 1protein that comprises an amino acid sequence that has at least about90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, and a functionally equivalent fragment thereof; or whereinthe structural nucleic acid sequence has at least 90% sequence identityto a nucleotide sequence selected from the group consisting of SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, and a functionally equivalentfragment thereof; regenerating from said transformed plant cell atransformed crop plant, wherein expression of the structural nucleicacid sequence in cells of the transformed crop plant causes reduction offunctional endogenous COP1 protein level; obtaining seeds from saidtransformed crop plant or progeny of said transformed crop plant; andplanting said seeds at a population density that is at least 10% higherthan normal.
 3. A transgenic plant comprising the recombinant DNAconstruct of claim
 1. 4. The transgenic plant of claim 3, wherein saidplant is selected from the group consisting of maize, wheat, rye,barley, oats, buckwheat, sorghum, rice, sunflower, canola, peas, beans,soybeans, cotton, linseed, cauliflower, asparagus, lettuce, tobaccomustard, sugar beet, potato, sweet potato, carrot, turnip, celery,tomato, egg plant, cucumber, squash, apple, apricot, peach, pear, plum,orange, blackberry, blueberry, strawberry, cranberry and lemon.
 5. Thetransgenic plant of claim 3, wherein said plant is a maize plant. 6.Progeny or seeds of the transgenic plant of claim 4, wherein the progenyor seeds comprise the recombinant DNA construct.
 7. Progeny or seeds ofthe transgenic plant of claim 5, wherein the progeny or seeds comprisethe recombinant DNA construct.
 8. A plant cell comprising therecombinant DNA construct of claim
 1. 9. The recombinant DNA constructof claim 1, wherein said promoter comprises a light-inducible promoter.10. The recombinant DNA construct of claim 1, wherein said promoter isselected from the group consisting of a cab promoter, an ATHB-2promoter, and a far red light inducible promoter.
 11. The recombinantDNA construct of claim 1, wherein said promoter comprises a cabpromoter.
 12. The recombinant DNA construct of claim 1, wherein the COP1nucleotide sequence encodes a polypeptide having an amino acid sequencethat has at least 95% sequence identity to the amino acid sequence ofSEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, ora functionally equivalent fragment thereof; or wherein said COP1nucleotide sequence has at least 95% sequence identity to a nucleotidesequence selected from the group consisting of SEQ ID NO:19, and SEQ IDNO:20 or a functionally equivalent fragment thereof.
 13. The recombinantDNA construct of claim 1, wherein the COP1 nucleotide sequence encodes apolypeptide having the amino acid sequence of SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, or a functionallyequivalent fragment thereof; or wherein said COP1 nucleotide sequencecomprises the nucleotide sequence of SEQ ID NO:19, SEQ ID NO:20, or afunctionally equivalent fragment thereof.
 14. The method of claim 2,wherein said constitutive photomorphogenesis 1 protein comprises aprotein binding domain and wherein said protein binding domain binds toa COP1 protein.
 15. The method of claim 2, wherein said structuralnucleic acid sequence is overexpressed in the cells of said crop plant.16. The method of claim 2, wherein the population density is at least40% higher than the average prevailing density for said crop in saidgrowing region.
 17. The method of claim 2, wherein the populationdensity is at least 70% higher than the average prevailing density forsaid crop in said growing region.
 18. The method of claim 2, wherein thepopulation density is at least 100% higher than the average prevailingdensity for said crop in said growing region.
 19. The method of claim 2,wherein said crop plant is selected from the group consisting of maize,wheat, rye, barley, oats, buckwheat, sorghum, rice, sunflower, canola,peas, beans, soybeans, cotton, linseed, cauliflower, asparagus, lettuce,tobacco mustard, sugar beet, potato, sweet potato, carrot, turnip,celery, tomato, egg plant, cucumber, squash, apple, apricot, peach,pear, plum, orange, blackberry, blueberry, strawberry, cranberry andlemon.
 20. The method of claim 2, wherein said crop plant is a maizeplant.